Upon cultivation with vegetable oils such as soybean oil or rapeseed oil, Pseudozyma aphidis produces extracellular biosurfactants, named mannosylerythritol lipids (MELs) (Rau et al., 2005). These are considered very promising due to their high fermentation yields (over 100 g/l) and excellent surface-active and potential pharmaceutical properties. The fermentation product consists of a complex mix of residual free fatty acids and four types of MELs with different degrees of acetylation. These four MEL types are classified as MEL-A, -B, -C and -D, and are hereinafter referred to as formulae (Ia), (Ib), (Ic) and (Id) respectively.

Said MEL types have respectively two acetyl (Ac) groups on C4′ and C6′, one acetyl group on C6′, one acetyl group on C4′ and no acetyl groups at all on the mannose moiety. MEL-A with the highest acetylation degree represents 50-60% of the total MELs, MEL-B 12-17%, MEL-C 20-33% and MEL-D only 5-10%.
Acetylated products are known to be hydrolytically unstable, resulting in the unwanted release of acetic acid with a penetrating odor as a consequence and hereby compromising product quality and stability, especially for applications such as in food or cosmetics. An additional advantage of the deacetylated MELs is the reduced complexity of the fermentation product, which simplifies its purification.
Chemical hydrolysis of (acetyl)esters is widely applied, resulting in a carboxylic acid (acetic acid) and an alcohol. To catalyze these reactions a high pH is required. Usually a base such as KOH or NaOH is used. However, alkaline hydrolysis is an aggressive method and would in this case hydrolyze all ester bonds present in the MEL molecule, thereby removing its fatty acyl chains which form the hydrophobic tail of the surfactant.
To address this issue, enzymatic deacetylation can be used as a mild alternative. Under carefully selected process conditions, enzymes can catalyze specific reactions. Fukuoka et al. (2011) reported the enzymatic deacetylation of mannosylerythritol lipids using a lipase and a water-containing organic solvent. In particular, Fukuoka discloses the enzymatic deacetylation of a diastereomer of MEL-B produced by the yeast Pseudozyma tsukubaensis using the lipase from Candida antarctica immobilized on acrylic resin, i.e. Novozym® 435. The solvent used was 90% ethanol (thus containing 10% water). The reaction took 7 days resulting in a conversion of >99% starting from 50 g/l MEL-B. The calculated productivity (grams of substrate conversion per liter per day) of this reaction, based on Fukuoka's results, is approximately 7 g/l·d using 25 g/l of Novozym® 435 with an activity of 10,000 PLU/g. Such long reaction times, i.e. seven days or more, are typical for enzymatic deacetylation reactions of glycolipid biosurfactants (Fukuoka et al., 2011; Hu et al., 2003). For the conventional MEL-A (as opposed to the diastereomer), Fukuoka et al., 2011 only partially obtained a MEL which was more hydrophilic, namely MEL-C instead of MEL-D. As detailed in the examples that follow herein after, a repetition of the experiments by Fukuoka et al., using 90% ethanol (+10% water) was performed. However, this resulted in a very low and slow conversion (only 18 conversion after 24 h-72 h). On the other hand, a parallel experiment in accordance with the current invention was carried out where water was omitted from the reaction solvent. Absolute ethanol was used here resulting in a vast improvement of the deacetylation of the conventional MEL-B (91% conversion after 24 h).
These data further support that for enzymatic reactions, the reaction conditions and solvents are crucial, and preferably do not cause the denaturation of the enzymes, since these are in general very expensive and thus preferably reused after the reaction. Polar solvents like water, and methanol are known to have a detrimental effect on free lipases and immobilized lipases such as Novozym® 435, especially at their optimal working temperatures >45° C. (José et al., 2011)
It was thus an object of the present invention to provide a more suitable reaction medium for the deacetylation of mannosylerythritol lipids, wherein the enzyme doesn't suffer major activity loss and can be reused. Furthermore, it was an object of the present invention to reduce the reaction time, to make the process economically feasible.